Mechanism-based inactivator

A general kinetic scheme for mechanism-based inactivators is outlined as follows ... 

An evaluation of potential mechanism-based inactivators requires the hilfillment of the following criteria (/) irreversible, active site-directed... 

Criteria very similar to those for mechanism-based inactivators apply for the evaluation of pseudoirreversible inhibitors. The kinetics for pseudoirreversible inactivators are as follows ... 

A significant difference between pseudoirreversible inhibitors and mechanism-based inactivators is the reversibiUty of the inactivation. A complete evaluation of the mechanism involved would require evidence not only for the covalent enzyme-inhibitor complex, but also for its decomposition products and its rate of reactivation. It is often difficult to identify the active site amino acid residue covalently linked to the inhibitor because of the instabiUty of the complex. 

Vaillancourt FH, G Labbe, NM Drouin, PD Fortin, LD Eltis (2002) The mechanism-based inactivation of 2,3-dihydroxybiphenyl 1,2-dioxygenase by catecholic substrates. J Biol Chem 277 2019-2027. 

Wakselman, M. Xie, J. Mazaleyrat, J.-P. Boggetto, N. Vilain, A. C. Montagne, J.-J. Reboud-Ravaux, M. New mechanism-based inactivators of trypsin-like proteinases. Selective inactivation of urokinase by functionalized cyclopeptides incorporating a sulfoniomethyl-substituted mera-aminobenzoic acid residue. J. Med. Chem. 1993, 36, 1539-1547. 

P Schmeidlin-Ren, DJ Edwards, ME Fitzsimmons, K He, KS Lown, PM Woster, A Rahman, KE Thummel, JM Fisher, PF Hollenberg, PB Watkins. Mechanisms of enhanced oral availability of CYP3A4 substrates by grapefruit constituents decreased enterocyte CYP3A4 concentration and mechanism-based inactivation by furanocoumarins. Drug Metab Dispos 25 1228-1233, 1997. 

Because these mechanism-based inactivators rely on the chemistry of the enzyme active site, they are often highly selective for the target enzyme and thus provide the specificity required for use as drug molecules. 

Figure 8.2 Mechanisms of irreversible enzyme inactivation. (A) Nonspecific affinity labeling, (B) quiescent affinity labeling, and (C) mechanism-based inactivation.

Owing to their reliance on enzyme catalysis to generate the inhibitory species, mechanism-based inactivators can be very specific for the target enzyme, or at the very least, highly selective for a family of enzymes that catalyze a common reac-... 

Only when k5 is rate-limiting can we equate kma with k5. Likewise K, has a complex form for mechanism-based inactivation ... 

Despite the mechanistic differences in the definitions of kimcl and A) between quiescent affinity labels and mechanism-based inactivators, the dependence of kohs on [/] is the same for both mechanism. Hence we cannot determine whether or not a compound is acting as a mechanism-based inhibitor, based merely on this two-step kinetic behavior. However, there is a set of distinguishing features of mechanism-based inactivation that are experimentally testable. Compounds that display all of these features can be safely defined as mechanism-based inactivators. 

Several authors have discussed specific criteria for designating a compound as a mechanism-based inactivator. Abeles and Maycock (1976) and Walsh (1978) were among the first to set out specific experimental tests for mechanism-based inactivation. More recently Silverman (1988, 1992, 1995) has described a comprehensive set of seven distinguishing features that mechanism-based inactivators must display. These are described here. 

Figure 8.9 Reaction progress curve in the presence of a mechanism-based inactivator when a second aliquot of enzyme is added to the reaction solution. The reaction is allowed to reach a plateau before a second, equal concentration aliquot of enzyme is added at the indicated time point. Note that the rate of inactivation for this second aliquot of enzyme is the same as that seen in the initial progress curve.

As described above, mechanism-based inactivation conforms to a two-step reaction and should therefore display saturation behavior. The value of should be a rectangular hyperbolic function of [/]. This was described in detail above in Section 8.1. 

Because mechanism-based inactivators behave as alternative substrates for the enzyme, they must bind in the enzyme active site. Binding of a mechanism-based inactivator is therefore mutually exclusive with binding of the cognate substrate of the normal enzymatic reaction (we say cognate substrate here because for bisubstrate reactions, the mechanism-based inactivator could be competitive with one substrate and noncompetitive or uncompetitive with the other substrate of the reaction, depending on the details of the reaction mechanism). Thus, as the substrate concentration is increased, the observed rate of inactivation should decrease (Figure 8.10) as... 

Figure 8.10 Substrate protection of an enzyme against irreversible inactivation by a mechanism-based inactivator. The data points in this plot are fitted to Equation (8.7).

Note that in some cases one may follow the time course of covalent E-A formation by equilibrium binding methods (e.g., LC/MS, HPLC, NMR, radioligand incorporation, or spectroscopic methods) rather than by activity measurements. In these cases substrate should also be able to protect the enzyme from inactivation according to Equation (8.7). Likewise a reversible competitive inhibitor should protect the enzyme from covalent modification by a mechanism-based inactivator. In this case the terms. S and Ku in Equation (8.7) would be replaced by [7r] and K respectively, where these terms refer to the concentration and dissociation constant for the reversible inhibitor. 

Mechanism-based inactivation results in formation of a covalent adduct between the active inhibitor and the enzyme, or between the active inhibitor and a substrate or cofactor molecule. If the mechanism involves covalent modification of the enzyme, then one should not be able to demonstrate a recovery of enzymatic activity after dialysis, gel filtration, ultrafiltration, or large dilution, as described in Chapters 5 to 7. Additionally, if the inactivation is covalent, denaturation of the enzyme should fail to release the inhibitory molecule into solution. If a radiolabeled version of the inactivator is available, one should be able to demonstrate irreversible association of radioactivity with the enzyme molecule even after denaturation and separation by gel filtration, and so on. In favorable cases one should likewise be able to demonstrate covalent association of the inhibitor with the enzyme by a combination of tryptic digestion and LC/MS methods. 

Because mechanism-based inactivation depends on enzyme catalysis, there cannot be more than one molecule of inactivator bound to the enzyme active site. Thus formation of the covalent E-A species cannot result in a stoichiometry of inactivator to enzyme of greater than 1 1. In the case of multimeric enzymes, however, it may not be necessary to covalently modify all of the enzyme active sites within the multi-mer in order to effect total inactivation of the enzyme. In this situation one may observe a stoichiometry of less that 1 1. Under no circumstances, however, can a mechanism-based inactivator display a stoichiometry of greater than 1 1 with the enzyme. 

In the kinetic scheme of Figure 8.2C, we see that once the active species is formed, it can go on to inactivate the enzyme directly or be released into solution. If the active species formed is a good affinity label (i.e., is highly electrophilic), there is a chance that this species will rebind and inactivate the enzyme as an affinity label. To be classified as a mechanism-based inactivator, the active species must be demonstrated to directly inactivate the enzyme while still bound, without reliance on dissociation from the EA complex. 

A second test for buildup of a free inactivator is to measure product formation in the presence of an excess of compound until the progress curve reaches a plateau, and to then add a second aliquot of enzyme. As described earlier in this section, the addition of a second aliquot of enzyme should result in renewed product formation, which will wane with time as the new molecules of enzyme are inactivated. The rate of inactivation of the second aliquot of enzyme (measured as kobs) should be the same as that of the first aliquot of enzyme in the experiment, if the compound is functioning as a true mechanism-based inactivator. If, instead, inactivation is due to buildup of an inhibitory species, then the second value of lcohs should be greater than the first value. This experiment can also be performed by preincubating the enzyme with compound and initiating the reaction with cognate substrate, as... 

The partitioning of the activated inhibitor between direct covalent inactivation of the enzyme and release into solution is an important issue for mechanism-based inactivators. The partition ratio is of value as a quantitative measure of inactivation efficiency, as described above. This value is also important in assessing the suitability of a compound as a drug for clinical use. If the partition ratio is high, this means that a significant proportion of the activated inhibitor molecules is not sequestered as a covalent adduct with the target enzyme but instead is released into solution. Once released, the compound can diffuse away to covalently modify other proteins within the cell, tissue, or systemic circulation. This could then lead to the same types of potential clinical liabilities that were discussed earlier in this chapter in the context of affinity labels, and would therefore erode the potential therapeutic index for such a compound. 

Potential Clinical Advantages of Mechanism-Based Inactivators... 

---